During complement activation the 74-77 amino acid anaphylatoxins C3a, C4a and C5a are released. They are potent inflammatory mediators, inducing cellular degranulation, smooth muscle contraction, arachidonic acid metabolism, cytokine release, cellular chemotaxis (Reviewed in Gerard, C., and Gerard, N. P. (1994) Annu. Rev. Immunol. 12, 775-808; Hugli, T. E. (1984) Springer Semin. Immunopathol. 7, 193-219; Bitter-Suermann, D. (1988) in The Complement System, Ed. by K. Rother & G. Till, Springer Verlag, Heidelberg 367-395) and have been implicated in the pathogenesis of a number of inflammatory diseases (Vogt, W. (1986) Complement 3, 177-188; Morgan, B. P. (1994) European J Clin Investigation 24, 219-228). Studies have demonstrated the presence of a C3a receptor (C3a-R) on guinea pig platelets, rat mast cells, human neutrophils, eosinophils and platelets (Bitter-Suermann, D. (1988) in The Complement System, Ed. by K. Rother & G. Till, Springer Verlag, Heidelberg 367-395). A single class of high affinity C3a binding sites has been characterized on human neutrophils and differentiated U937 cells (Klos, A., Bank, S., Gietz, C., Bautsch, W., Kohl, J., Burg, M., and Kretzschmar, T. (1992) Biochemistry 31, 11274-11282). Competition binding and functional desensitization studies are consistent with the presence of a receptor for C3a which is distinct from the C5a-R (Bitter-Suermann, D. (1988) in The Complement System, Ed. by K. Rother & G. Till, Springer Verlag, Heidelberg 367-395; Klos, A., Bank, S., Gietz, C., Bautsch, W., Kohl, J., Burg, M., and Kretzschmar, T. (1992) Biochemistry 31, 11274-11282). However, there is evidence that C3a and C4a may bind to the same receptor as the two anaphylatoxins cross desensitize guinea pig ileal tissue (Hugli, T. E. (1984) Springer Semin. Immunopathol. 7, 193-219; Bitter-Suermann, D. (1988) in The Complement System, Ed. by K. Rother & G. Till, Springer Verlag, Heidelberg 367-395), although other investigators using guinea pig macrophages indicate that there may be separate receptors (Murakami, Y., Yamamoto, T., Imamichi, T., Nagasawa, S. (1993) Immuol. Lett. 36, 301-304). Functional activity of the C3a-R is sensitive to pertussis toxin, consistent with the binding site being composed of a GPCR (Klos, A., Bank, S., Gietz, C., Bautsch, W., Kohl, J., Burg, M., and Kretzschmar, T. (1992) Biochemistry 31, 11274-11282).
A complete understanding of the role of C3a in the pathogenesis of the inflammatory response has been hampered by the lack of ligand characterization of the cloned receptor. The present invention provides methods of using and functional characterization of human C3a receptor. This receptor was recently cloned from an HL-60 library by low-stringency screening with a fMetdeuPhe receptor probe. (Roglic, A., Prossnitz, E. R., et al. (1996) Biochimica et Biophysica Acta 1305, 3943). The report of this receptor contained no characterization of the receptor as a C3a receptor. Functional data on this important, useful feature was lacking in the report. It was characterized as an orphan receptor (AZ3B). Mouse L cells expressing AZ3B failed to bind and respond to the agonists examined, although C3a was not tested (Roglic, A., Prossnitz, E. R., et al. (1996) Biochimica et Biophysica Acta 1305, 39-43). The present invention provides important uses for the C3a receptor and compounds that agonize and antagonize C3a receptor function.
Clarification of this receptor as a C3a receptor and methods and compounds exploiting this important characterization are provided herein. Clearly, there is a need for factors that mediate inflammation and their roles in dysfunction and disease. There is a need, therefore, for identification and further characterization of such factors that mediate inflammation, and which can play a role in preventing, ameliorating or correcting dysfunctions or diseases.
The polypeptide used in the methods of the present invention has the conserved complement receptor residues, and have amino acid sequence homology to known complement receptors.